Aug 29, 1984 - sources plus an amino acid supplement (20 ,ug/ml) as re- quired. Kanamycin ..... of urinary salicyclic, salicyluric and gentisic acids. J. Chroma-.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1985,
Vol. 49, No. 4
p. 761-764
0099-2240/85/040761-04$02.00/0 Copyright © 1985, American Society for Microbiology
Plasmid-Borne TnS Insertion Mutation Resulting in Accumulation of Gentisate from Salicylate D. J. MONTICELLO,t D. BAKKER, M. SCHELL, AND W. R. FINNERTY* Department of Microbiology, University of Georgia, Athens, Georgia 30602 Received 29 August 1984/Accepted 3 January 1985
Plasmid-borne TnS insertion mutants of a Pseudomonas species which accumulated 2,5-dihydroxybenzoate (gentisate) following growth on 2-hydroxybenzoate (salicylate) were obtained from a pool of mutants that were unable to grow on naphthalene. One such mutant was characterized further. The ability of this mutant to oxidize gentisate was 100-fold less than the ability of a Nah+ Sal' strain harboring the unmutagenized plasmid, although both strains oxidized and grew on salicylate. These bacteria were presumably able to metabolize salicylate via catechol, since they possessed an inducible, plasmid-encoded catechol 2,3-dioxygenase. Our results suggest that there is an alternate, plasmid-encoded route of salicylate degradation via gentisate and that some plasmid-associated relationship between this pathway and naphthalene oxidation exists.
Plasmid DNA was extracted from cells and visualized on 0.8% agarose gels by using the method of Kado and Liu (9). Purified plasmid DNA was prepared by CsCl centrifugation of cell lysates (11). DNA transfer from agarose gels to nitrocellulose filters, 32P nick translation of pBR322::TnS DNA, and hybridizations were performed as described by Davis et al. (5). Naphthalene and salicylate metabolites were separated and identified by thin-layer chromatography (TLC) as previously described (13). Products were obtained by acidifying the culture medium to pH 3.0 with 6 N HCl, extracting with ethyl acetate, and drying under reduced pressure. Absorption spectra were determined with a Cary model 219 scanning spectrophotometer. Salicylate metabolites were analyzed by using a Varian model 5000 high-pressure liquid chromatograph and a Micropak MCH10 column (30 cm by 4 mm). The solvent system consisted of acetonitrile (120 ml), water (280 ml), and concentrated formic acid (0.4 ml) and was used at a flow rate of 2 ml/min. In 14C-labeling experiments cells were grown to stationary phase (2 days) in 100 ml of 0. 1% salicylate medium supplemented with [7-14C]salicylate (20 ,XCi/flask; specific activity, 53.8 mCi/mmol; New England Nuclear Corp.). The acidified spent medium was extracted with ethyl acetate. Cathechol 1,2-dioxygenase and catechol 2,3-dioxygenase were assayed by the method of Barnsley (1). Substrate-dependent 02 utilization by whole cells was determined by using a Clark oxygen electrode.
Salicylate (2-hydroxybenzoate) is a key intermediate in the plasmid-encoded catabolism of naphthalene by pseudomonads (Fig. 1), and gentisate (2,5-dihydroxybenzoate) and its alkyl-substituted analogs are similarly important in the plasmid-encoded degradation of m-cresol, 2,5-xylenol, 3,5-xylenol, and related compounds (2, 8). Although the conversion of salicylate to gentisate has been documented in eucaryotic systems (15), this conversion is not generally observed in bacteria (7, 14). We generated a pool of plasmidborne TnS insertion mutants from a Pseudomonas species which was able to grow on naphthalene (Nah+) or salicylate (Sal') by using the strategy described by Yen and Gunsalus (16). Initially, we intended to use these mutants to examine the interrelationship of the Nah+ and Sal' phenotypes, both of which are mediated by a 72-kilobase plasmid designated pDBT2 (11). We identified Nah- Sal' Tn5 insertion mutants which accumulate novel metabolites following growth on salicylate. In this report we describe the production of these mutants, the characterization of particular Nah- mutants, and the identification of one of the salicylate metabolites as gentisate. MATERIALS AND METHODS The isolation and characterization of the soil isolate which we used (designated isolate DBT2 and identified as Pseudomonas alcaligenes) are described in the accompanying paper (11). The origins, preparation, and properties of the other strains used are shown in Table 1. Cells were grown on medium which contained 4 g of K2HPO4 per liter, 4 g of Na2HPO4 per liter, 2 g of (NH4)2SO4 per liter, 0.2 g of MgSO4 * 7H20 per liter, 0.001 g of CaCl2 * 2H20 per liter, and 0.001 g of FeSO4 * 7H20 per liter, was adjusted to pH 7.0 with HCl, and was supplemented with carbon and energy sources plus an amino acid supplement (20 ,ug/ml) as required. Kanamycin resistance (Kmr), gentamicin resistance (Gmr), streptomycin resistance (Smr), and nalidixic acid resistance (Nalr) were tested at concentrations of 100 ,ug/ml on plates containing 0.8% nutrient broth-0.5% yeast extract medium (NBYE medium) supplemented with 1.6% agar.
RESULTS
Preparation and characterization of TnS insertion mutants. A Pseudomonas putida AC-10 variant containing a chromosomal Tn5 insertion was constructed by filter mating (4) of a Nalr P. putida AC-10 variant and Escherichia coli 1830 containing "suicide" plasmid pJB4JI (3). Although all Nalr Kmr exconjugants were Gmr and retained plasmid pJB4JI (as visualized on agarose gels), subsequent overnight growth with novobiocin (25 ,ug/ml) in NBYE medium and screening for Kmr Gms yielded Kmr P. putida AC-10 colonies which did not harbor plasmid pJB4JI (strain DM1011), as determined by gel electrophoresis. Conjugation of isolate DBT2 with one of these strain DM1011 colonies and selection for Sal' Kmr yielded strain DM1211, which harbored pDBT2.
* Corresponding author. t Present address: Enzyme Process Development Laboratory, Miles Laboratories, Inc., Elkhart, IN 46514.
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